1. Field of the Invention
The invention relates to the field of apparatus and methods for optogenetic treatment of blindness including retinitis pigmentosa.
2. Description of the Prior Art
Retinitis pigmentosa (RP) refers to disorders characterized by degeneration of photoreceptors in the eye which hinders visual ability by nonfunctional neuronal activation and transmission of signals to the cortex. The prevalence of this disease is at least one million individuals. The disease is most often inherited as an autosomal recessive trait with 50 to 60% of cases having this form of inheritance. However, since not all RP-causing genes have been discovered, if a person chooses to get genetically tested, there is about a 50 percent chance that the disease causing gene will be identified. Although there is no definitive nonsurgical therapy available for RP, numerous claims of therapeutic triumphs have been made in the United States and abroad within the last decade. Though an experimental ‘rescue’ of retinas by treating eyes with certain stem cells has been shown to preserve visual function in mice that were genetically predisposed to degenerative diseases of the retina, most of the current clinical treatments are primarily focused on slowing down the progression of the disease, as there is no cure that can stop the disease; there is no therapy that can restore any vision lost due to this disease.
The invasive surgical procedure for partial restoration of vision is by retinal implants/transplants. The subretinal implants are positioned in the area of the retina where the rod and cone photoreceptor cells reside, between the pigmented epithelium and the bipolar cells. The epiretinal implants are placed in the area of the retinal ganglion cells. The subretinal implants are composed of a 50-100 μm thin plate which is 2-3 mm in diameter and on this plate there is an array of microphotodiodes and microelectrodes. Light is detected by the microphotodiodes and then transformed into electrical currents which are delivered to neurons by the microelectrodes. The subretinal implants are surgically placed within the eye by entering the vitreous humor of the eye and making a scleral incision to gain access to the subretinal space. The disadvantages with using the subretinal implants also include the fact that the device undergoes damage over a period of time. Another disadvantage is that one microphotodiode will not be able to produce a current sufficient enough to stimulate adjacent neurons with the use of ambient light. In epiretinal implants, no light sensitive elements are used on the implant itself. The implant is placed in the same area where the retinal ganglion cells reside and the device functions by stimulating the axons of the retinal ganglion cells in response to input obtained from a camera that can be placed outside of the eye or within an intraocular lens. The use of an epiretinal implant requires that any visual information which is obtained by the camera will be translated into a spatiotemporal pattern of electrical stimuli. There are certain disadvantages to using this approach, primarily due to surgically implanting the device. It is difficult to place the device in the epiretinal space and if damage occurs during the implantation procedure, then cellular proliferation can occur. Furthermore, a disordered stimulation pattern can result while using epiretinal implants due to the fact that the electrical stimulation can stimulate both the axons and cell bodies of the ganglion cells. Currently, studies have been done on blind individuals with the use of epiretinal implants eliciting a response where the individuals were able to see patterns of light. However, the results have not been able to produce any object recognition in individuals.
Another invasive technique for treating retinitis pigmentosa involves the use of retinal transplants. Animal studies have been done which have shown some success in utilizing this approach. In one study, sheets of fetal retina with fetal pigment epithelium were transplanted into the subretinal space of rats. The experiment showed successful functioning of the retina through the recording of visually evoked responses that corresponded to the area where the transplant was placed. Furthermore, studies have been done on human subjects in which neural retinal progenitor cell layers with retinal pigment epithelium were transplanted into the eyes of a small number of patients having retinitis pigmentosa and age-related macular degeneration. Though results of the study showed that the visual acuity is improved in some of the patients, critical issues exists in transplantation of a larger number of tissue sheets into the retina.
Besides being highly invasive in nature, all the above methods for restoration of vision are based on very non-specific cellular activation and have low spatial resolution, and hence have not been very successful in restoration of vision.
The optogenetic treatment is based on a very recent phenomenon where chemically identical neurons can be activated by blue light with high temporal precision by introducing a light-activated molecular channel, named channelrhodopsin-2 (ChR2), into specific groups of cells by genetic targeting. This eliminates the highly challenging requirement of placing electrodes inside every single neuron of a chemically identified group of cells. This method also has several advantages over electrical stimulation such as cellular specificity. and noninvasiveness. Since ChR2 is a non-selective cation channel, light-induced activation of ChR2 results in depolarization of only those neurons that express ChR2. Selective activation of neurons by ms-pulsed blue light has been demonstrated in cell culture, brain slices, as well as in small animals. This optogenetic activation method is very attractive and practical as it only requires light of very low intensity (few mW/mm2) that can be delivered from a lamp with a bandpass filter or small laser diode. Though this technique eliminates the highly challenging requirement of placing electrode arrays and it has advantages over electrical stimulation such as cellular specificity, higher resolution and non-invasiveness, transfection of a specific cellular layer in the retina requires efficient delivery of genes. Therefore, the only in vivo attempt for restoration of vision in blind mice models involves delivery of the ChR2 gene by the viral method. Besides the increasing concern about use of viruses, a ubiquitous CMV promoter was used, which is known to target non-specifically the whole retina. The recent promoter specific study targeting bipolar cells used in-utero electroporation, but it is not applicable for in-vivo applications. Further, since the spatiotemporal pattern of action potentials generated by retinal ganglion neurons ultimately determines the raw input from the eyes to the brain, specific targeting of retinal ganglion cells should provide a better and efficient method for restoration of vision.
Light-assisted activation of selected groups of neurons has been made possible with high temporal precision by introducing a light-activated molecular channel, named channelrhodopsin-2 (ChR2) into specific groups of cells by genetic targeting. This optogenetic activation method is very attractive and practical as it only requires light of very low intensity. Though this technique eliminates the highly challenging requirement of placing electrode arrays and has advantages over electrical stimulation such as cellular specificity and non-invasiveness, this optogenetic method requires transfection of specific cellular layer in retina. The viral method of delivery of the ChR2 coding gene has been primarily a concern for successful application of this technique. Related known prior art includes Miller G (2006) Shining new light on neural circuits. Science 314: 1674-1676. Bi, A., Cui, Ma, Y.-P., Olshevskaya, E., Pu, M., Dizhoor, A. M., and Pan, Z.-H. (2006). Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration Neuron 50: 23-33. Lagali P S, Balya D, Awatramani G B, et. al. (2008). “Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration”. Nat. Neurosci. 11 (6): 667-75.
Therefore, there is a need of a systematic method for nonviral delivery of the ChR2 gene into retinal ganglion cells of the adult retina so as to create visually evoked potentials in the visual cortex. However, the two reported studies for restoration of vision in blind mice models involve delivery of ChR2 gene either by viral methods or by in-utero electroporation which is not applicable for in vivo applications. Further, no studies were found describing targeting of retinal ganglion cells specifically and effective behavioral improvement.